The translational goal in Project 4 is to identify and bring forward into clinical trials one or two lead compounds that will suppress c-Myc transcription by targeting the NM23-H2-DNA complex. This proposal builds upon progress from the last grant period and will further test hypothesis that the NM23-H2-DNA complex is critically involved in transcriptional activation of c-Myc and c-Myc is a critical factor in colon cancer. The specific aims of the proposal are (1) structural characterization of the G-quadruplex-drug complexes in the c-Myc promoter, (2) to establish in vitro biochemical and cell-based screens to identify small molecules that inhibit binding of NM23-H2 to the G-quadruplex in the silencer element of the c-Myc promoter, (3) to discover and optimize G-quadruplex- and NM23-H2-interactive compounds using computer- aided drug design and structure-based and virtual-screening approaches, (4) In vivo evaluation and subsequent preclinical development, and (5) to file an IND for Phase I clinical trial and overall clinical development program. High-field NMR will be used to characterize the drug-G-quadruplex complex, molecular modeling will be used to identify new lead compounds, and fluorescence resonance energy transfer (FRET) and double-filter methods will be used for identification of in vitro biochemical hits. A luciferase-based high-throughput screen has already been used to identify hits, and subsequent assays will be carried out in matched cell lines genetically engineered to provide proof of principle that the mechanism of action is as proposed. Structure-based approaches will be used to aid in lead optimization. Following lead optimization, an IND will be filed and the clinical development program will be based upon discoveries from specific aims 1-4. Immunohistochemistry will be used to guide patient selection and phase I and II clinical trial plans will be designed to determine whether the new agents will help patients with advanced colorectal cancer. The long-term goal of this research is to develop novel target-directed drugs for treatment of colorectal cancer.